Recent studies have shown that both plant and animal bacterial innate immunity share many features in common in response to pathogen infection. The Arabidopsis/Pseudomonas syringae pathosystem is a highly tractable model system to study the molecular basis of bacterial plant innate immunity. The complete genome sequence of both interacting partners has greatly facilitated many molecular approaches including;bioinformatic, genetic, biochemical, cell biological and structural biological studies. A hallmark feature of the bacterial innate immune system in Arabidopsis thaliana is the surveillance of bacterial effector proteins delivered to the plant cell via a type III secretion system by cognate resistance proteins. In our laboratory, we have focused on the RPS2 disease resistance-signaling pathway that specifically recognizes the bacterial effector protein, AvrRpt2. The RPS2 gene encodes for CC/NB/LRR protein and represents a member of one of the subclasses of the "superfamily" of NB/LRR disease resistance proteins found in all plants. The goals of this project are elucidate the molecular events that specify the recognition of bacterial effector proteins and the biochemical and cellular signaling events that determine bacterial innate immunity. In aim 1, we will identify and characterize the amino acid recognition cleavage site of the AvrRpt2 protease. We propose to employ a molecular genetic strategy that will allow us to define and characterize the AvrRpt2 protease cleavage site in AvrRpt2 and RIN4. The identification and characterization of this site will provide insight in the biochemical mechanism of proteolysis and may aid in the identification of addition protein targets in the Arabidopsis genome for this important virulence effector protein. In aim 2, we will purify the AvrRpt2 and RIN4 Proteins for Biochemical Activity Studies. The development of these assays will be critical for the development of the biochemical isolation of the proposed eukaryotic factor that is essential for protease activity described in aim 3. In aim 3, we propose to identify and characterize the putative eukaryotic factor required for AvrRpt2 protease activity by concomitant biochemical and genetic experimental strategies. In aim 4, we will define the molecular basis of RIN4 negative regulation by defining which protein domains of RIN4 are involved in the repression of RPS2 activation and which domains of RPS2 are interacting with RIN4 by utilizing deletion mutagenesis strategies in conjunction with co-immunoprecipitation experiments. Finally in aim 5, we propose to genetically identify mutations that compromise the induction of the Rps2-specified disease resistance Pathway. The genetic dissection of the Rps2 pathway coupled with the identification of RPS2-interacting proteins will put us in a strong position to uncover the molecular events controlling the Rps2 resistance-signaling pathway.